Bottom Line:
DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics.Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro.These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

ABSTRACTT cells primed in the gut-draining mesenteric lymph nodes (mLN) are imprinted to express alpha4beta7-integrin and chemokine receptor CCR9, thereby enabling lymphocytes to migrate to the small intestine. In vitro activation by intestinal dendritic cells (DC) or addition of retinoic acid (RA) is sufficient to instruct expression of these gut-homing molecules. We report that in vivo stroma cells, but not DC, allow the mLN to induce the generation of gut tropism. Peripheral LN (pLN) transplanted into the gut mesenteries fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and prime T cells. DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics. Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro. These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

fig1: Transplantation of LN fragments yields chimeric LN constituted by donor-derived stromal cells and recipient-derived hematopoietic cells. (A) 8 wk after transplantation, Tx-pLN and Tx-mLN could be identified in situ by expression of EGFP. (B) To demonstrate low autofluorescence in endogenous LN, an excised Tx-pLN was placed besides the endogenous pLN. (C and D) Tx-pLN were analyzed by fluorescence microscopy for EGFP expression (green). Expression of gp38 (C) and ER-TR7 (D) is depicted in red. Comparable results were obtained for Tx-mLN (not depicted). Bars, 20 μm. (E) Cells were isolated from Tx-LN, and EGFP expression by stroma cells, DC, B, and T cells was analyzed by flow cytometry (data are pooled from four mice analyzed in two experiments). Error bars represent SD.

Mentions:
To study the role of LN stroma cells in the generation of gut-homing effector T cells, mLN were excised from WT mice and replaced by either pLN or mLN fragments isolated from EGFP mice. 8 wk after surgery, transplanted LN (Tx-LN) could be identified by their EGFP expression (Fig. 1, A and B). In line with previous observations, we noted that Tx-LN displayed normal tissue architecture and cellular composition (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080039/DC1) (14) and were connected to gut-draining afferent and LN efferent lymphatics (not depicted) (15). Fluorescent microscopy on T cell zones in Tx-LN revealed that EGFP expression colocalized with gp38 (Fig. 1 C) and ER-TR7 (Fig. 1 D), which are both expressed by LN stroma cells, whereas EGFP+ cells showed no overlap with anti-B220 and anti-CD3 staining (not depicted). Similar results were obtained when cells isolated by collagenase digestion of Tx-LN were analyzed by flow cytometry. Few B and T cells, as well as DC, expressed EGFP (Fig. 1 E). Flow cytometry of CD45−gp38+ stroma cells revealed almost 50% EGFP− cells (Fig. 1 E). Because immunohistology showed that in the T cell zones of Tx-LN the vast majority of stroma cells expressed EGFP, this indicates that donor- and host-derived stroma cells are unevenly distributed in the various compartments of the transplant. In conclusion, a major population of donor-derived nonhematopoietic stroma cells survived in the Tx-LN, whereas the vast majority of hematopoietic cells were constituted by host cells.

fig1: Transplantation of LN fragments yields chimeric LN constituted by donor-derived stromal cells and recipient-derived hematopoietic cells. (A) 8 wk after transplantation, Tx-pLN and Tx-mLN could be identified in situ by expression of EGFP. (B) To demonstrate low autofluorescence in endogenous LN, an excised Tx-pLN was placed besides the endogenous pLN. (C and D) Tx-pLN were analyzed by fluorescence microscopy for EGFP expression (green). Expression of gp38 (C) and ER-TR7 (D) is depicted in red. Comparable results were obtained for Tx-mLN (not depicted). Bars, 20 μm. (E) Cells were isolated from Tx-LN, and EGFP expression by stroma cells, DC, B, and T cells was analyzed by flow cytometry (data are pooled from four mice analyzed in two experiments). Error bars represent SD.

Mentions:
To study the role of LN stroma cells in the generation of gut-homing effector T cells, mLN were excised from WT mice and replaced by either pLN or mLN fragments isolated from EGFP mice. 8 wk after surgery, transplanted LN (Tx-LN) could be identified by their EGFP expression (Fig. 1, A and B). In line with previous observations, we noted that Tx-LN displayed normal tissue architecture and cellular composition (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080039/DC1) (14) and were connected to gut-draining afferent and LN efferent lymphatics (not depicted) (15). Fluorescent microscopy on T cell zones in Tx-LN revealed that EGFP expression colocalized with gp38 (Fig. 1 C) and ER-TR7 (Fig. 1 D), which are both expressed by LN stroma cells, whereas EGFP+ cells showed no overlap with anti-B220 and anti-CD3 staining (not depicted). Similar results were obtained when cells isolated by collagenase digestion of Tx-LN were analyzed by flow cytometry. Few B and T cells, as well as DC, expressed EGFP (Fig. 1 E). Flow cytometry of CD45−gp38+ stroma cells revealed almost 50% EGFP− cells (Fig. 1 E). Because immunohistology showed that in the T cell zones of Tx-LN the vast majority of stroma cells expressed EGFP, this indicates that donor- and host-derived stroma cells are unevenly distributed in the various compartments of the transplant. In conclusion, a major population of donor-derived nonhematopoietic stroma cells survived in the Tx-LN, whereas the vast majority of hematopoietic cells were constituted by host cells.

Bottom Line:
DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics.Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro.These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

ABSTRACTT cells primed in the gut-draining mesenteric lymph nodes (mLN) are imprinted to express alpha4beta7-integrin and chemokine receptor CCR9, thereby enabling lymphocytes to migrate to the small intestine. In vitro activation by intestinal dendritic cells (DC) or addition of retinoic acid (RA) is sufficient to instruct expression of these gut-homing molecules. We report that in vivo stroma cells, but not DC, allow the mLN to induce the generation of gut tropism. Peripheral LN (pLN) transplanted into the gut mesenteries fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and prime T cells. DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics. Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro. These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.